QC Report

	general
Report generated at	2024-11-08 20:47:40
Title	Humanliver_ATAC-seq
Description	Humanliver_hg38_ATAC-seq_encode_pipeline_Mary
Pipeline version	v2.2.3
Pipeline type	atac
Genome	hg38
Aligner	bowtie2
Sequencing endedness	{'rep1': {'paired_end': True}, 'rep2': {'paired_end': True}}
Peak caller	macs2

Alignment quality metrics

SAMstat (raw unfiltered BAM)

	rep1	rep2
Total Reads	193675982	300654650
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	191593429	296451244
Mapped Reads (QC-failed)	0	0
% Mapped Reads	98.9	98.6
Paired Reads	193675982	300654650
Paired Reads (QC-failed)	0	0
Read1	96837991	150327325
Read1 (QC-failed)	0	0
Read2	96837991	150327325
Read2 (QC-failed)	0	0
Properly Paired Reads	189518268	293213598
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	97.89999999999999	97.5
With itself	190875260	294977500
With itself (QC-failed)	0	0
Singletons	718169	1473744
Singletons (QC-failed)	0	0
% Singleton	0.4	0.5
Diff. Chroms	27454	44867
Diff. Chroms (QC-failed)	0	0

Marking duplicates (filtered BAM)

	rep1	rep2
Unpaired Reads	0	0
Paired Reads	78364751	126687267
Unmapped Reads	0	0
Unpaired Duplicate Reads	0	0
Paired Duplicate Reads	29240399	24698587
Paired Optical Duplicate Reads	0	0
% Duplicate Reads	37.3132	19.4957

Filtered with samtools flag 1804 (samtools view -F 1804):

read unmapped (0x4)
mate unmapped (0x8, for paired-end)
not primary alignment (0x100)
read fails platform/vendor quality checks (0x200)
read is PCR or optical duplicate (0x400)

Fraction of mitochondrial reads (unfiltered BAM)

	rep1	rep2
Rn = Number of Non-mitochondrial Reads	128802940	257568789
Rm = Number of Mitochondrial Reads	68799582	42695616
Rm/(Rn+Rm) = Frac. of mitochondrial reads	0.34817157849837566	0.14219339784880594

SAMstat (filtered/deduped BAM)

	rep1	rep2
Total Reads	95205822	201514074
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	95205822	201514074
Mapped Reads (QC-failed)	0	0
% Mapped Reads	100.0	100.0
Paired Reads	95205822	201514074
Paired Reads (QC-failed)	0	0
Read1	47602911	100757037
Read1 (QC-failed)	0	0
Read2	47602911	100757037
Read2 (QC-failed)	0	0
Properly Paired Reads	95205822	201514074
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	100.0	100.0
With itself	95205822	201514074
With itself (QC-failed)	0	0
Singletons	0	0
Singletons (QC-failed)	0	0
% Singleton	0.0	0.0
Diff. Chroms	0	0
Diff. Chroms (QC-failed)	0	0

Filtered and duplicates are removed. Subsampling with atac.subsample_reads is not done in alignment steps. Nodup BAM is converted into a BED type (TAGALIGN) later and then TAGALIGN is subsampled with such parameter in the peak-calling step.

Fragment length statistics (filtered/deduped BAM)

	rep1	rep2
Fraction of reads in NFR	0.5819935897641559	0.5207237190578763
Fraction of reads in NFR (QC pass)	True	True
Fraction of reads in NFR (QC reason)	OK	OK
NFR / mono-nuc reads	2.211479147110098	1.8753482820014797
NFR / mono-nuc reads (QC pass)	False	False
NFR / mono-nuc reads (QC reason)	out of range [2.5, inf]	out of range [2.5, inf]
Presence of NFR peak	True	True
Presence of Mono-Nuc peak	True	True
Presence of Di-Nuc peak	True	True

Open chromatin assays show distinct fragment length enrichments, as the cut sites are only in open chromatin and not in nucleosomes. As such, peaks representing different n-nucleosomal (ex mono-nucleosomal, di-nucleosomal) fragment lengths will arise. Good libraries will show these peaks in a fragment length distribution and will show specific peak ratios.

NFR: Nucleosome free region

Sequence quality metrics (filtered/deduped BAM)

Open chromatin assays are known to have significant GC bias. Please take this into consideration as necessary.

Annotated genomic region enrichment

	rep1	rep2
Fraction of Reads in universal DHS regions	0.3766133650944162	0.3920419275529113
Fraction of Reads in blacklist regions	0.0019270355125971183	0.0015658807036971522
Fraction of Reads in promoter regions	0.15665509405506733	0.1631528723894491
Fraction of Reads in enhancer regions	0.3113037771996759	0.31495788725903084

Signal to noise can be assessed by considering whether reads are falling into known open regions (such as DHS regions) or not. A high fraction of reads should fall into the universal (across cell type) DHS set. A small fraction should fall into the blacklist regions. A high set (though not all) should fall into the promoter regions. A high set (though not all) should fall into the enhancer regions. The promoter regions should not take up all reads, as it is known that there is a bias for promoters in open chromatin assays.

Library complexity quality metrics

Library complexity (filtered non-mito BAM)

	rep1	rep2
Total Fragments	54025915	112851930
Distinct Fragments	47790957	101513762
Positions with Two Read	4968796	9056123
NRF = Distinct/Total	0.884593	0.899531
PBC1 = OneRead/Distinct	0.884336	0.900864
PBC2 = OneRead/TwoRead	8.505732	10.098147

Mitochondrial reads are filtered out by default. The non-redundant fraction (NRF) is the fraction of non-redundant mapped reads in a dataset; it is the ratio between the number of positions in the genome that uniquely mapped reads map to and the total number of uniquely mappable reads. The NRF should be > 0.8. The PBC1 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with AT LEAST one read pair. PBC1 is the primary measure, and the PBC1 should be close to 1. Provisionally 0-0.5 is severe bottlenecking, 0.5-0.8 is moderate bottlenecking, 0.8-0.9 is mild bottlenecking, and 0.9-1.0 is no bottlenecking. The PBC2 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with EXACTLY two read pairs. The PBC2 should be significantly greater than 1.

Fragment: read for a single-ended dataset, pair of reads for a paired-ended dataset
NRF: non redundant fraction
PBC1: PCR Bottleneck coefficient 1
PBC2: PCR Bottleneck coefficient 2
PBC1 is the primary measure. Provisionally

0-0.5 is severe bottlenecking
0.5-0.8 is moderate bottlenecking
0.8-0.9 is mild bottlenecking
0.9-1.0 is no bottlenecking

Replication quality metrics

IDR (Irreproducible Discovery Rate) plots

Reproducibility QC and peak detection statistics

	overlap	idr
Nt	223290	128816
N1	188490	103876
N2	229809	148380
Np	243527	159958
N optimal	243527	159958
N conservative	223290	128816
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.0906310179587084	1.24175568252391
Self Consistency Ratio	1.2192105681999046	1.4284339019600292
Reproducibility Test	pass	pass

Reproducibility QC

N1: Replicate 1 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep1 reads)
N2: Replicate 2 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep2 reads)
Ni: Replicate i self-consistent peaks (comparing two pseudoreplicates generated by subsampling RepX reads)
Nt: True Replicate consistent peaks (comparing true replicates Rep1 vs Rep2)
Np: Pooled-pseudoreplicate consistent peaks (comparing two pseudoreplicates generated by subsampling pooled reads from Rep1 and Rep2)
Self-consistency Ratio: max(N1,N2) / min (N1,N2)
Rescue Ratio: max(Np,Nt) / min (Np,Nt)
Reproducibility Test: If Self-consistency Ratio >2 AND Rescue Ratio > 2, then 'Fail' else 'Pass'

Number of raw peaks

	rep1	rep2
Number of peaks	299245	299390

The number of peaks is capped at 300000
Peaks are called from macs2 with p-val threshold 0.01

Peak calling statistics

Peak region size

	rep1	rep2	idr_opt	overlap_opt
Min size	150.0	150.0	150.0	150.0
25 percentile	211.0	241.0	466.0	333.0
50 percentile (median)	334.0	409.0	715.0	547.0
75 percentile	602.0	728.0	1056.0	886.0
Max size	2914.0	3409.0	3607.0	3607.0
Mean	463.3262811408712	542.6293830789272	799.7239837957463	662.6261153794035

Enrichment / Signal-to-noise ratio

TSS enrichment (filtered/deduped BAM)

	rep1	rep2
TSS enrichment	23.294805448649445	20.962438980086663

Open chromatin assays should show enrichment in open chromatin sites, such as TSS's. An average TSS enrichment in human (hg19) is above 6. A strong TSS enrichment is above 10. For other references please see https://www.encodeproject.org/atac-seq/

Jensen-Shannon distance (filtered/deduped BAM)

	rep1	rep2
AUC	0.2263420399191345	0.24240738277199259
Synthetic AUC	0.49626988097767083	0.4974896859719107
X-intercept	0.10509648372212048	0.09602589426375682
Synthetic X-intercept	0.0	0.0
Elbow Point	0.7097531328220589	0.7516613753228786
Synthetic Elbow Point	0.501843246571	0.49871701474440583
Synthetic JS Distance	0.3929482746498775	0.3810945307670934

Peak enrichment

Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

	rep1	rep2	rep1-pr1	rep2-pr1	rep1-pr2	rep2-pr2	pooled	pooled-pr1	pooled-pr2
Fraction of Reads in Peaks	0.3025505730101254	0.32557167694401334	0.29556460747611407	0.3176803391143753	0.3002228645265594	0.31652913053139037	0.3215049657472244	0.3155033214826508	0.3170344979769674

FRiP for overlap peaks

	rep1_vs_rep2	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.29834259917643	0.26856582363208836	0.30710027231150117	0.30705710411815457

FRiP for IDR peaks

	rep1_vs_rep2	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.24981365927682855	0.22027253753452178	0.2697370159862879	0.27180949470270777

For macs2 raw peaks:

repX: Peak from true replicate X
repX-prY: Peak from Yth pseudoreplicates from replicate X
pooled: Peak from pooled true replicates (pool of rep1, rep2, ...)
pooled-pr1: Peak from 1st pooled pseudo replicate (pool of rep1-pr1, rep2-pr1, ...)
pooled-pr2: Peak from 2nd pooled pseudo replicate (pool of rep1-pr2, rep2-pr2, ...)

For overlap/IDR peaks:

repX_vs_repY: Comparing two peaks from true replicates X and Y
repX-pr1_vs_repX-pr2: Comparing two peaks from both pseudoreplicates from replicate X
pooled-pr1_vs_pooled-pr2: Comparing two peaks from 1st and 2nd pooled pseudo replicates

Other quality metrics

Comparison to Roadmap DNase

This bar chart shows the correlation between the Roadmap DNase samples to your sample, when the signal in the universal DNase peak region sets are compared. The closer the sample is in signal distribution in the regions to your sample, the higher the correlation.